Abstract: To control or even eradicate a tumor, an effective immunotherapy requires the clonal expansion of tumor- specific cytotoxic T lymphocytes, a non-exhaustive tumor microenvironment and the correct homing of effector cells within the tumor. I propose to exploit the novel functions of unnatural chemokine conjugates as mono or combination therapies to satisfy the aforementioned criteria. The interaction of chemokines with their respective receptors will be explored as a means for targeted antigen delivery. Specifically, tumor-associated MUC1(Tn) glycopeptide antigen will be conjugated to sortase-ready chemokines to afford structurally defined epitope vaccines that can target antigen presenting cells (APCs) for efficient antigen delivery, presentation, and cross-presentation. The introduction of chemically synthesized tumor-associated carbohydrate antigens through a sortase-mediated transpeptidase reaction is critical for achieving immune response towards posttranslationally modified tumor epitopes that cannot be easily introduced by genetic approaches. I further aspire to promote the effective homing of effector cells in a non-exhaustive tumor microenvironment using a chemokine delivery and monitoring platform based on the specificity of camelid-derived single domain antibody fragments (VHHs) that recognize the immunological checkpoint PD-L1. The presence of a sortase recognition motif engineered at the chemokine C-terminus will enable site-specific installation of multifunctional click handles in a sortase-catalyzed reaction, followed by the formation of an unnatural C-C fusion with VHHs that recognize and block the immune checkpoint PD-L1. A similar chemical strategy allows the installation of isotopes suitable for positron emission tomography (PET) to monitor the biodistribution of chemokines and their fusions in living animals. Fusions that show tumor localization and retain chemotactic function will be examined for their ability to attract or repel responsive leukocytes in vivo via immuno-PET, using an isotopically labeled ?CD8 VHH. The successful completion of the following aims will provide tools immediately useful to explore posttranslational tumor markers for cancer vaccination and leukocyte recruitment to the tumor microenvironment (TME) for successful cancer immunotherapies. Aim 1. Engineer and recombinantly express sortase-ready chemokines. Aim 2. Chemokine-MUC1(Tn) conjugates for antigen delivery and immunization. Aim 3. Investigate the in vivo biodistribution and chemotactic function of chemokine-VHH fusions (CVFs) with PD-L1 blocking specificity.